Bacillus subtilis can be transformed by exogenous DNA when in a physiological state known as competence. Competent cells are able to bind double-stranded DNA, fragment the DNA on the cell surface, and transport a single strand across the cell envelope. The long-term objective of this project is to understand these processes on the molecular level. A dozen genes required for transformation, and their protein products, have been characterized. The interactions among these proteins and their roles in DNA binding and transport will be investigated by genetic and biochemical methods. These will include mutagenesis of individual proteins followed by the search for allele- specific suppression, the analysis of protein/protein interactions using surface plasmon resonance, cross-linking and immunoprecipitation, and determination of the accessibility of a known competence DNA binding protein to exogenous molecular probes of varying sizes in mutants lacking other competence proteins. The fate of transforming DNA will be characterized in mutants lacking known competence proteins, and the proteins contacted by transforming DNA during binding and uptake will be characterized using a novel DNA-protein cross-linking approach. An in vitro DNA transport system will be developed using membrane vesicles. Additional genes required for transformation will also be isolated and characterized. The possibility that the type 4 pilin-like ComG proteins interact with and remodel the cell wall will also be investigated, as will the effect of the ComGA protein on replication and segregation of chromosomes in competent cells.